Instruction Manual
Catalog #600886 (single kit)
#600887 (10-pack kit)
Revision C
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CONTENTS
Materials Provided.............................................................................................................................. 1
Storage Conditions .............................................................................................................................. 1
Additional Materials Required .......................................................................................................... 1
Notices to Purchaser ........................................................................................................................... 1
Introduction......................................................................................................................................... 2
Features of Kit Components.................................................................................................. 2
Fluorescence Monitoring in Real-Time................................................................................. 3
Preprotocol Considerations................................................................................................................ 5
RNA Isolation........................................................................................................................ 5
Quantitative PCR Human Reference Total RNA .................................................................. 5
RT-PCR Primer Concentration.............................................................................................. 6
Preventing Sample Contamination ........................................................................................ 6
Magnesium Chloride ............................................................................................................. 6
Reference Dye ....................................................................................................................... 7
Reaction Preparation ............................................................................................................. 7
Temperature and Duration of cDNA Synthesis Reaction...................................................... 8
Recommended Control Reactions ......................................................................................... 8
Data Acquisition with a Spectrofluorometric Thermal Cycler.............................................. 8
Protocol ................................................................................................................................................ 9
Preparing the Reactions......................................................................................................... 9
RT-PCR Cycling Program................................................................................................... 10
Dissociation Program .......................................................................................................... 11
Troubleshooting ................................................................................................................................ 12
References .......................................................................................................................................... 13
Endnotes............................................................................................................................................. 13
MSDS Information............................................................................................................................ 13
Quick-Reference Protocol ................................................................................................................ 15
Brilliant III Ultra-Fast SYBR Green QRT-PCR Master Mix
MATERIALS PROVIDED
Catalog #600886 (single kit), #600887 (10-pack kit)
Materials Provided Quantity a,b
2 Brilliant III Ultra-Fast SYBR Green QRT-PCR Master Mix 2 2 ml
RT/RNase Block 400 l
100 mM DTT 100 l
Reference Dye , 1 mM
c
100 l
a
Sufficient reagents are provided for four hundred, 20-l QRT-PCR reactions.
b
Quantities listed are for a single kit. For 10-pack kits, each item is provided at 10 times the listed quantity.
c
The reference dye is light sensitive and should be kept away from light whenever possible.
STORAGE CONDITIONS
All Components: Store at 20C upon receipt. After thawing, the 2 master may be stored at 4C
for up to three months or returned to 20C for long term storage
Note The reference dye and master mix are light sensitive and should be kept away from light
whenever possible.
NOTICES TO PURCHASER
Notice to Purchaser: Limited License
Purchase of this product includes an immunity from suit under patents specified in the product insert
to use only the amount purchased for the purchasers own internal research. No other patent rights
are conveyed expressly, by implication, or by estoppel. Further information on purchasing licenses
may be obtained by contacting the Director of Licensing, Applied Biosystems, 850 Lincoln Centre
Drive, Foster City, California 94404, USA.
The kit includes the components necessary to carry out cDNA synthesis and
PCR amplification in one tube and one buffer.* The single-step master mix
format is ideal for most high-throughput QPCR applications where it is not
necessary to archive cDNA.
RT/RNase Block
The reverse transcriptase (RT) provided in the kit is a Moloney-based RT
specifically formulated for the Stratagene Brilliant III Ultra-Fast kits. This
RT performs optimally at a reaction temperature of 50C when used in
1-step QRT-PCR with the Brilliant III master mix. It is stringently quality-
controlled to verify the absence of nuclease contaminants that adversely
affect cDNA synthesis and to ensure sensitive and reproducible performance
in QRT-PCR experiments with a broad range of RNA template amounts and
a variety of RNA targets that vary in size, abundance, and GC-content. The
RNase block, provided in the same tube, serves as a safeguard against
contaminating RNases.
DTT
A separate tube of 100 mM DTT is provided with the kit. Adding DTT to
the reactions improves RT performance for more challenging targets.
In Figure 1, the Brilliant III Ultra-Fast SYBR Green QRT-PCR master mix
was used in reactions containing serially diluted RNA template and a
no-template control reaction (NTC) to amplify the GAPDH target. In the
amplification plot (top panel) the reactions containing template show a
significant increase in fluorescence with Ct values ranging from 15 to 36.
The Ct values obtained in the amplification plot are used to generate the
standard curve in the bottom left panel. In the dissociation/melt curve
(bottom, right), PCR samples were subjected to a stepwise increase in
temperature from 60C to 95C with fluorescence measurements taken
throughout this range. The first derivative of fluorescence is then plotted
versus temperature. As the temperature increases, the amplification products
in each tube melt according to their composition. In Figure 1, the melt curve
shows fluorescence peaks centered at 83C, which correspond to the desired
product in the reactions. If primer-dimer or nonspecific products had been
made during the amplification step, they would generally melt at a different
temperature (defined as the Tm) than the desired products.
Magnesium Chloride
The optimal MgCl2 concentration promotes maximal amplification of the
specific target amplicon with minimal nonspecific products and primer-
dimer formation. High levels of the Mg2+ ion tend to favor the formation of
nonspecific dsDNA, including primer-dimers. Therefore, when a SYBR
Green-based QPCR assay is being optimized, the MgCl2 levels should be as
low as possible, as long as the efficiency of amplification of the specific
target is not compromised (typically between 1.5 and 2.5 mM MgCl2). The
Brilliant III SYBR Green QRT-PCR master mix contains MgCl2 at a
concentration of 2.5 mM (in the 1 solution), which is suitable for most
targets. The concentration may be increased, if desired, by adding a small
amount of concentrated MgCl2 to the 1 experimental reaction at the time of
set up.
Reaction Preparation
* The diluted reference dye, if stored in a light-protected tube at 4C, can be used within the
day for setting up additional assays.
No-RT Control
We recommend performing no-RT control reactions for each experimental
sample by omitting the RT/RNase block from the reaction. The no-RT
control is expected to generate no signal if there is no amplification of
genomic DNA. No signal indicates that the RNA preparation is free of
contaminating genomic DNA or that the primers are specific for the cDNA.
See Preventing Genomic DNA Contamination in RNA Isolation.
Endogenous Control
Consider performing an endogenous control reaction to normalize variation
in the amount of RNA template across samples. See Reference 2 for
guidelines on the use of endogenous controls for QPCR.
3. Mix the reagents well without creating bubbles, then distribute the
mixture to individual PCR reaction tubes. Keep the reactions on ice.
5. Gently mix the reactions without creating bubbles, then centrifuge the
reactions briefly.
6. Place the reactions in the instrument. Based on the instrument you are
using, select the appropriate PCR program from the tables below. Set
the instrument to detect and report fluorescence at each cycle during the
60C annealing/extension step.
* The exact duration needs to be optimized for each target. Challenging templates (e.g. GC-rich RNA or RNA with extensive
secondary structure) generally require longer denaturation and annealing/extension times than low-complexity templates.
ENDNOTES
Primer Express is a registered trademark of The Perkin-Elmer Corporation.
SYBR is a registered trademark of Molecular Probes, Inc.
MSDS INFORMATION
The Material Safety Data Sheet (MSDS) information for Stratagene products is provided on the web at
http://www.genomics.agilent.com. MSDS documents are not included with product shipments.
QUICK-REFERENCE PROTOCOL
Prior to setting up the reactions, thaw the 2 QRT-PCR master mix and store on ice. Following initial
thawing of the master mix, the unused portion may be stored at 4C for up to three months or
returned to 20C for long term storage.
SYBR Green I dye (present in the master mix) is light-sensitive; solutions containing the
master mix should be protected from light whenever possible.
1. If using the reference dye, dilute the provided dye with nuclease-free PCR-grade H2O. For the
ABI StepOnePlus instrument or the ABI 7900HT Fast instrument, dilute the dye 1:50 (for a final
concentration of 300 nM in the reactions). For the Stratagene Mx3000P or Mx3005P
instrument or the ABI 7500 Fast instrument, dilute the dye 1:500 (for a final concentration of 30
nM in the reactions). Keep all solutions containing the reference dye protected from light.
2. Prepare the experimental reactions by adding the following components in order. Prepare a
single reagent mixture for multiple reactions using multiples of each component listed below.
Keep the reagent mixture on ice.
Reagent Mixture
Nuclease-free PCR-grade H2O to bring the final volume to 20 l (including experimental RNA)
10 l of 2 SYBR Green QRT-PCR master mix
x l of upstream primer (optimized concentration)
x l of downstream primer (optimized concentration)
0.2 l of 100 mM DTT
0.3 l of diluted reference dye from step 1 (optional)
1 l of RT/RNase block
3. Mix the reagents well without creating bubbles, then distribute the mixture to individual PCR
reaction tubes. Keep the reactions on ice.
5. Gently mix the reactions without creating bubbles, then centrifuge the reactions briefly.
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6. Place the reactions in the instrument. Based on the instrument you are using, select the
appropriate PCR program from the tables below. Set the instrument to detect and report
fluorescence at each cycle during the 60C annealing/extension step.
7. For your specific instrument, follow the manufacturers guidelines for generating dissociation
curves.
* The exact duration needs to be optimized for each target. Challenging templates (e.g. GC-rich RNA or RNA with extensive
secondary structure) generally require longer denaturation and annealing/extension times than low-complexity templates.
16